Zebrafish reporter lines reveal in vivo signaling pathway activities involved in pancreatic cancer

Marco Schiavone,Alessandro Casari,Elena Rampazzo,Giusy Battilana,Steven D Leach,Francesco Argenton,Luca Persano,Natascia Tiso,Shu Liu,Enrico Moro

doi:10.1242/dmm.014969

Abstract

Pancreatic adenocarcinoma, one of the worst malignancies of the exocrine pancreas, is a solid tumor with increasing incidence and mortality in industrialized countries. This condition is usually driven by oncogenic KRAS point mutations and evolves into a highly aggressive metastatic carcinoma due to secondary gene mutations and unbalanced expression of genes involved in the specific signaling pathways. To examine in vivo the effects of KRASG12D during pancreatic cancer progression and time correlation with cancer signaling pathway activities, we have generated a zebrafish model of pancreatic adenocarcinoma in which eGFP-KRASG12D expression was specifically driven to the pancreatic tissue by using the GAL4/UAS conditional expression system. Outcrossing the inducible oncogenic KRASG12D line with transgenic zebrafish reporters, harboring specific signaling responsive elements of transcriptional effectors, we were able to follow TGFβ, Notch, Bmp and Shh activities during tumor development. Zebrafish transgenic lines expressing eGFP-KRASG12D showed normal exocrine pancreas development until 3 weeks post fertilization (wpf). From 4 to 24 wpf we observed several degrees of acinar lesions, characterized by an increase in mesenchymal cells and mixed acinar/ductal features, followed by progressive bowel and liver infiltrations and, finally, highly aggressive carcinoma. Moreover, live imaging analysis of the exocrine pancreatic tissue revealed an increasing number of KRAS-positive cells and progressive activation of TGFβ and Notch pathways. Increase in TGFβ, following KRASG12D activation, was confirmed in a concomitant model of medulloblastoma (MDB). Notch and Shh signaling activities during tumor onset were different between MDB and pancreatic adenocarcinoma, indicating a tissue-specific regulation of cell signaling pathways. Moreover, our results show that a living model of pancreatic adenocarcinoma joined with cell signaling reporters is a suitable tool for describing in vivo the signaling cascades and molecular mechanisms involved in tumor development and a potential platform to screen for novel oncostatic drugs.

Highlights

Pancreatic adenocarcinoma is one of the most aggressive cancers in industrial countries, and its incidence and mortality is still increasing
Upon comparison of pancreatic adenocarcinoma with concomitant ptf1a-induced medulloblastoma in the cerebellum, they conclude that transforming growth factor β (TGFβ), Sonic Hedgehog (Shh) and Notch are involved in both cancers at different stages of carcinogenesis
The identification of mechanisms regulating the hallmarks of this aggressive disease and of candidate molecules (Notch, TGFβ and Shh ligands and their effectors) with the potential to interfere with cancer progression is an important step towards new therapies for pancreatic adenocarcinoma

Summary

Introduction

Pancreatic adenocarcinoma is one of the most aggressive cancers in industrial countries, and its incidence and mortality is still increasing. Shh plays a major role in stem-cell proliferation (Wu et al, 2010); the Wnt pathway is involved in cell proliferation and differentiation (Yang, 2012); Notch is responsible for stem-cell maintenance (Kwon et al, 2012); and TGFβ controls cell and tissue homeostasis, favoring cell apoptosis by cross-talking with other pathways such as p53 (Elston and Inman, 2012). Shh and Notch pathways seem to be involved in tumor onset, together with genomic instability, whereas Wnt and TGFβ appear activated in cancer progression by eliciting cell migration or neo-angiogenesis through reciprocal cross-talk or by interactions with other pathways (McCleary-Wheeler et al, 2012). It has a tumor suppressor function at early tumor stages, whereas at later stages it mediates oncogenic effects (Heldin and Moustakas, 2012; McCleary-Wheeler et al, 2012; Truty and Urrutia, 2007)

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